The invention relates to novel insulin activity (IA) proteins and nucleic acids. The Invention further relates to the use of the IA proteins in the treatment of Insulin-related disorders such as type 1 diabetes and type 2 diabetes.
Insulin is a hormone that plays a major role in the regulation of growth and metabolism in vertebrates. A deficiency of insulin is the most important factor in diabetic disease states. Absence of insulin leads to severe metabolic disorders resulting from the failure to normally metabolize carbohydrate, fat and proteins at a normal rate. These disorders include, for example diabetes mellitus (DM), a complex chronic metabolic disorder. Diabetes mellitus is characterized in two broad groups based on clinical manifestations, namely the non-insulin-dependent diabetes (NIDDM) or maturity onset form, also known as Type 2 and the insulin-dependent diabetes (IDDM) or juvenile onset form, also known as Type 1. In the general population, diabetes mellitus occurs with a prevalence of approximately 1%, with one fourth of these being the Type1. In its most fully expressed clinical form, diabetes mellitus manifests itself as a series of hormone-induced metabolic abnormalities which eventually lead to serious, long-term and debilitating complications such as glucosuria, ketonuria, growth arrest, and negative nitrogen balance. These conditions can ultimately lead to death. Familial hyperproinsulinemla is a genetic disorder characterized by a marked increase in serum proinsulin-like molecules. The cause of this disease is an amino acid substitution which results in incomplete cleavage of proinsulin by the proteases which form insulin.
Type 1 diabetes arises for example, when patients lack beta-cells producing insulin in their pancreatic glands or when the produced insulin is inactive due to mutation(s). Type 2 diabetes occurs in patients with an impaired beta cell function. Type 1 diabetic patients are currently treated with insulin, while the majority of type 2 diabetic patients are treated either with sulfonylureas that stimulate beta cell function or with agents that enhance the tissue sensitivity of the patients towards insulin (e.g., metformin) or with insulin.
Today, insulin administration to diabetic patients is the primary therapeutic means for controlling the disease. In the treatment of diabetes mellitus, many varieties of insulin preparations have been suggested and used. Some of the preparations are fast acting and other preparations have more or less prolonged actions. Such a prolonged action may be obtained by administering the insulin as a suspension of insulin crystal which can be obtained by crystallization of insulin in the presence of zinc (such as LENTE; Novo Terapeutisk Laboratorium) or by crystallization of insulin in the presence of zinc and protamine (such as NPH-insulin).
The human insulin monomer, a 6000 dalton protein, is composed of two chains, the 21 amino acid A-chain and the 30 amino acid B-chain. Insulin is synthesized in pancreatic beta cells located within the islets of Langerhans as a precursor form that is post-translationally processed to the mature two polypeptide chain active hormone. In the biologically active human insulin, the A and B chains are linked with one another via two cysteine bridges, and a further cysteine bridge occurs within the A-chain. The following cysteine residues are linked with one another in human insulin: A6-A-11, A7-B7, and A20B-19 (the letters A and B stand for the amino acid chain, and the numbers for the position of the cysteine residues counted from the amino to carboxyl end of each chain; see FIG. 1).
Based on the functional analysis of insulins from various species and several insulin analogs or mutants, some pertinent properties of insulin have emerged with respect to the amino acid sequence (see also FIG. 1): biologically active insulin has three disulfide bonds; B1-Phe is present in all known mammalian insulins; A1-Gly; a terminal tripeptide sequence (A19-21) Tyr-Cys-Asn (removal of A21-Asn by carboxypeptidase digestion results in a loss of activity of greater than 90%); an invariant sequence at B24-26, Phe-Phe-Tyr; B12-Val is highly conserved; A2-A3, Ile-Val is highly conserved; B5-His and B22-Arg are invariant in insulins of high potency; invariant surface residues (highly conserved) A1-Gly, A4-Glu, A5-Gln, A7-Cys, A19-Tyr, A21-Asn, and B7-Cys.
To this end, variants of insulin sequences, applications, production procedures and assays are known;
see for example U.S. Pat. No. 4,421,685 (reports process for producing insulin); U.S. Pat. No. 4,992,417 (reports superactive insulin analogues); U.S. Pat. No. 5,008,241 (reports insulin analogs characterized by amino acid residue substitutions of N21 in the A-chain, with a resulting improvement in stability of insulin solutions at acidic pH levels); U.S. Pat. No. 5,506,202 (reports preparation and use of insulin derivatives comprising various amino acid substitutions); U.S. Pat. No. 5,514,646 (reports analogs of human insulin modified at position 29 of the B-chain that have modified physico-chemical and pharmacokinetic properties and are useful in the treatment of hyperglycemia); U.S. Pat. No. 5,559,094(reports analogs of human insulin containing an aspartic acid at position 1 of the B-chain and U.S. Pat. No. 5,618,913 (reports rapid acting human insulin analogues having amino acid residue substitution and less tendency to self-associate into dimers, tetramers, hexamers or polymers); U.S. Pat. No. 5,621,073 (reports a process for purification of insulin or an insulin acetylated at position A9); U.S. Pat. No. 5,663,291 (reports a process for obtaining insulin having correctly linked cysteine bridges from a corresponding proinsulin amino acid chain); U.S. Pat. No. 5,700,662 (reports process for preparing insulin analogs comprising a modification of position 29 of the B-chain); U.S. Pat. No. 6,034,054 (reports a monomeric insulin analog formulation stabilized against aggregation); all of which are expressly incorporated by reference and further by Marki et al. [Hoppe Seylets Z. Phsiol. Chem. 360(11):1619-32 (1979)]; Hu et al. [Biochemistry 32(10):2631-5 (1993)]; Schwartz et al. [Proc. Natl. Acad. Sci. U.S.A. 84(18):6408-11(1987)]; Kitagawa et al. [Biochemistry 23(7):1405-13 (1984)]; Kobayashi et al. [Biochem. Biophs. Res. Commun. 107(1):329-36 (1982)]; Shoelson et al. [Biochemistry 31(6):1757-67 (1992]; and references cited therein, all of which are expressly incorporated as reference.
Human insulin in solution is known to exist in many molecular forms, such as the monomer, the dimer, the tetramer and the hexamer [Blundell et al., in Advances in Protein Chemistry, Academic Press, New York and London, Vol.26, pp279-330 (1972)], with the oligomer forms being favored at high insulin concentrations and the monomer being the active form of insulin. Insulin in the bloodstream is highly dilute, being 10xe2x88x9211 to 10xe2x88x928 M and is primarily in monomer form. The much more concentrated insulin stored in the beta cell of the pancreas and in the usual administerable solution is largely in the non-active hexamer form, as the well-known 2 zinc hexamer (see below). The delayed absorption phenomena [Binder, Diabetes Care 7(2):188-99 (1984)] is in some large part attributable to the required for the insulin to disassociate from hexamer, tetramer and dimer form into the active monomer form.
in the presence of zinc (Zn), natural human insulin associates to a 2 Zn-hexamer that functions as an allosteric protein. Phenolic ligands or certain salts are capable of inducing a conformational transition, resulting in the N-terminal 8 amino acids of the B-chain converting from an extended conformation to an Q-helix. This conformational state induced by phenolic ligands has been referred to as the R state and the apoinsulin form as the T state. The R state is more compact, less flexible, and the Zn exchange is retarded compared to the T state [Derewenda et al., Nature 338(6216):594-596 (1989)]. A stable intermediate state, T3R3 has been Identified that has one trimer in the T state and the other in the R state [Chothia et al., Nature 302(5908):500-505 (1983)]. The T3R3 state was formally known as the 4-Zn insulin structure which is induced by salts (e.g. chloride) or by limited amounts of phenolics [Kruger, et al., Biol. Chem. Hoppe-Seyler 371(8):669-673 (1990)].
The different allosteric states of insulin hexamer have been best characterized in the crystal state by X-ray crystallography [Bentley et al., Nature 261(5556):166-168 (1976); Smith and Dodson, Biopolymers 32(4):441-445 (1992)], in solution by proton NMR, circular dichroism [Renscheidt et al., Eur. J. Biochem. 142(1):7-14 (1984)], and visible absorption spectroscopy of Co2+ substituted insulins [Brader et al., Biochemistry 30(27):6636-6645 (1991)]. The biological significance of insulin allosterism has not been fully elucidated. The biologically active form of insulin is thought to be a monomer due to the dilute concentrations of insulin in the blood circulation [Frank et al., Diabetes 21(2):Suppl. 2:486-491 (1972)]. A receptor-mediated conformational change in the insulin conformation is thought to be required for binding [see Hua et al. Nature 354(6350):238-41 (1991); Bao et al. Proc. Natl. Acad. Sci. U.S.A. 94(7):2975-80 (1997)]. For the medicinal use of insulin, the T- greater than R conformations have important consequences. Most formulations of insulin are solutions or suspensions that contain phenolics that function as preservatives against bacterial contamination. The phenolic concentrations in insulin formulations are 2-10 times that necessary to induce the R conformation (Kruger et al., supra). The presence of phenolics in insulin formulations has also important consequences on the shelf-life stability [Brange et al., Pharm. Res. 9(6):715-726 (1992); Brange et al., Pharm. Res. 9(6):727-734 (1992); Brange and Langkjaer, Acta Pharm Nord.,4(3):149-158 (1992)] and possibly time action profile. Minimizing degradation of insulin formulations is extremely important in reducing undesirable side effects of insulin therapy.
The crystal structures of various recombinant insulin molecules are solved. The structures can be obtained from the Research Collaboratory for Structural Bioinformatics as entries into the Protein Data Bank (PDB). Insulin entries include wild type insulin (e.g., see PDB entries 1ZEH, 1ZNJ, 1ZNI, 1XDA, 4INS, and 9INS), insulin analogs or mutants (e.g., PDB entries 1IOH, 1IOG, 1B9E, 1BYZ, 1ZEI, 1A7F, 1HUI, 1LPH, and 1IZA), R6 (wild type or analog) insulin hexamers (e.g., PDB entries 5AIY, 4AIY, 3AIY, 2AIY, 1AIY, 1AI0, 1QIZ, 1QJ0, and 1QIY); insulin complexed with phenol (e.g., PDB entry 1ZEG), insulin complexed with 4hydroxybenzanide (e.g., PDB entry 1BEN), insulin (wild type or analog) complexed with Zn ions (e.g., PDB entries 1TYM, 1TYL, 7INS, 1TRZ, and 1IZB), and other variants (e.g., PDB entries 1MPJ, 2TCI, 3MTH, 6INS, and 2INS) and insulins in various pH solutions (e.g., PDB entries 1DPH, 1CPH, 1BPH, and 1 APH), all of which are expressly incorporated by reference.
When carrying out protein engineering to modify protein properties, usually one had to select from the following options: (i) site-specific mutagenesis and (ii) random mutagenesis of the nucleic acid encoding the protein, or (iii) post-translational chemical modifications. No matter which method of protein engineering is used, a key aspect is determining which amino acids to modify, because few choices will improve the properties of the protein. The available crystal structure of insulin allows a completely different approach by using computational protein design and the generation of more stable proteins or protein variants with an altered activity. Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellinga et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry 34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konol, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, WO98/47089, and U.S. Ser. No. 09/127,926 describe a system for protein design; both are expressly incorporated by reference.
A need still exists for proteins exhibiting both significant stability and insulin activity. Accordingly, it is an object of the invention to provide insulin activity (IA) proteins, nucleic acids and antibodies for the treatment of insulin-related disorders.
In accordance with the objects outlined above, the present invention provides non-naturally occurring insulin activity (IA) proteins (e.g. the proteins are not found in nature) comprising amino acid sequences that are less than about 98% identical to human insulin. The IA proteins have at least one altered biological property of an insulin protein; for example, the IA proteins will be more stable than insulin and bind to a cell comprising an insulin receptor. Thus, the invention provides IA proteins with amino acid sequences that have at least about 1-20 amino acid substitutions as compared to the human insulin sequence shown in FIG. 1B.
In a further aspect, the present invention provides non-naturally occurring IA protein conformers that have three dimensional backbone structures that substantially correspond to the three dimensional backbone structure of insulin. The amino acid sequence of the IA protein conformer and the amino acid sequence of insulin are less than about 98% Identical.
In an additional aspect, the changes are selected from the amino acid residues at positions selected from positions A2, A3, A5, A6, A7, A11, A15, A16, A19, A20, B2, B7, B11, B15, B18, B19, B22, and B24. In a preferred aspect of this embodiment, the changes are substitutions selected from the group of A7-S, A7-E, B2-E, B2-T, B4-Y, B7-Y, B4F, B7-E, and B7-D.
In a preferred aspect, the changes are selected from the amino acid residues at positions selected from positions B5 and B14. in a preferred aspect of this embodiments the changes are substitutions selected from the group of B5-F, B5-W, B14-F, B14-W, B14-Y, and B14-I.
In an additional aspect, the changes are selected from the amino acid residues at positions selected from positions A1, A10, A16, A17, A19, B1, B2,B4, B8, B11, B12, B14, B25, B26, B27, and B28. In a preferred aspect of this embodiment, the changes are substitutions selected from the group of A1-N, A10-Q, A16-Y, A17-Y, A19-F, B1-D, B2-K, B4-F, B8-L, B11-I, B12-R, B14W, B25-N, B26-F, B27-D, and B28-N.
In a further aspect, the invention provides recombinant nucleic acids encoding the non-naturally occurring IA proteins, expression vectors comprising the recombinant nucleic acids, and host cells comprising the recombinant nucleic acids and expression vectors.
In an additional aspect, the invention provides methods of producing the IA proteins of the invention comprising culturing host cells comprising the recombinant nucleic acids under conditions suitable for expression of the nucleic acids. The proteins may optionally be recovered. In a further aspect, the invention provides pharmaceutical compositions comprising an IA protein of the invention and a pharmaceutical carrier.
In an additional aspect, the invention provides methods for treating an insulin responsive condition comprising administering an IA protein of the invention to a patient. The insulin responsive condition includes a disorder of carbohydrate metabolism, type 1 diabetes, and type 2 diabetes.